Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.

Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: Insights into disease pathogenesis and troponin function

Mutations in a number of cardiac sarcomeric protein genes cause hypertrophic cardiomyopathy (HCM). Previous findings indicate that HCM-causing mutations associated with a truncated cardiac troponin T (TnT) and missense mutations in the β-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. In contrast, Lin et al. [Lin, D., Bobkova, A., Homsher, E. & Tobacman, L. S. (1996) J. Clin. Invest. 97, 2842–2848] characterized a TnT point mutation (Ile79Asn) and concluded that it might lead to hypercontractility and, thus, potentially a different mechanism for HCM pathogenesis. In this study, three HCM-causing cardiac TnT mutations (Ile79Asn, Arg92Gln, and ΔGlu160) were studied in a myotube expression system. Functional studies of wild-type and mutant transfected myotubes revealed that all three mutants decreased the calcium sensitivity of force production and that the two missense mutations (Ile79Asn and Arg92Gln) increased the unloaded shortening velocity nearly 2-fold. The data demonstrate that TnT can alter the rate of myosin cross-bridge detachment, and thus the troponin complex plays a greater role in modulating muscle contractile performance than was recognized previously. Furthermore, these data suggest that these TnT mutations may cause disease via an increased energetic load on the heart. This would represent a second paradigm for HCM pathogenesis.

Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth. p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1. Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein. Overexpression of CDK5 in yeast cells complemented most phenotypes associated with pho85Δ, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G1 progression defect. Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells. In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related. Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals.

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GH in early embryogenesis?

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages. In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose–response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40–50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GH. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription–PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Functional domains are specified to single-cell resolution in a Drosophila epithelium

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P{GAL4} enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary (“stellate”) cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an “identified cell” system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Functional antioxidant responsive elements

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE “core” sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE–binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis

Accumulated evidence attributes noncatalytic morphogenic activitie(s) to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for the Drosophila protein neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Here, we report impaired neurite outgrowth and loss of neurexin Iα mRNA under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth was partially rescued by addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restored both neurite extension and expression of neurexin Iα. Differential PCR display revealed expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system, is partially colocalized with AChE, and increases in rescued AS-ACHE cells. Our findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis.

Cloning and functional characterization of mouse IκBɛ

The biological activity of the transcription factor NF-κB is mainly controlled by the IκB proteins IκBα and IκBβ, which restrict NF-κB in the cytoplasm and enter the nucleus where they terminate NF-κB-dependent transcription. In this paper we describe the cloning and functional characterization of mouse IκBɛ. Mouse IκBɛ contains 6 ankyrin repeats required for its interaction with the Rel proteins and is expressed in different cell types where we found that it is up-regulated by NF-κB inducers, as is the case for IκBα and human IκBɛ. IκBɛ functions as a bona fide IκB protein by restricting Rel proteins in the cytoplasm and inhibiting their in vitro DNA binding activity. Surprisingly, IκBɛ did not inhibit transcription of genes regulated by the p50/p65 heterodimer efficiently, such as the human interferon-β gene. However, IκBɛ was a strong inhibitor of interleukin-8 expression, a gene known to be regulated by p65 homodimers. In addition, IκBɛ appears to function predominantly in the cytoplasm to sequester p65 homodimers, in contrast with the other two members of the family, IκBα and IκBβ, which also function in the nucleus to terminate NF-κB-dependent transcriptional activation.

Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.